Marchionni et al.

BMC Genomics 2013, 14:336

http://www.biomedcentral.com/1471-2164/14/336

METHODOLOGY ARTICLE Open Access

A simple and reproducible breast cancer

prognostic test
Luigi Marchionni1, Bahman Afsari4, Donald Geman3,4* and Jeffrey T Leek2,5*

Abstract
Background: A small number of prognostic and predictive tests based on gene expression are currently offered as
reference laboratory tests. In contrast to such success stories, a number of flaws and errors have recently been
identified in other genomic-based predictors and the success rate for developing clinically useful genomic
signatures is low. These errors have led to widespread concerns about the protocols for conducting and reporting
of computational research. As a result, a need has emerged for a template for reproducible development of
genomic signatures that incorporates full transparency, data sharing and statistical robustness.
Results: Here we present the first fully reproducible analysis of the data used to train and test MammaPrint, an
FDA-cleared prognostic test for breast cancer based on a 70-gene expression signature. We provide all the software
and documentation necessary for researchers to build and evaluate genomic classifiers based on these data. As an
example of the utility of this reproducible research resource, we develop a simple prognostic classifier that uses
only 16 genes from the MammaPrint signature and is equally accurate in predicting 5-year disease free survival.
Conclusions: Our study provides a prototypic example for reproducible development of computational algorithms
for learning prognostic biomarkers in the era of personalized medicine.
Keywords: Reproducible research, Gene expression analysis, Biomarkers, Top scoring pair, Prediction, Genomics,
Personalized medicine, Breast cancer, MammaPrint

Background According to a recent report [8] from the Institute of

Currently, a number of molecular-based prognostic and
predictive tests for breast cancer are offered as labora-
tory services for clinical use [1,2]. Such assays, which in-
clude MammaPrint [3], OncotypeDx [4], PAM50 Breast
Cancer Intrinsic Subtype Classifier [5], MapQuant Dx [6]
and Theros Breast Cancer Index [7], are implemented
by providing multiple gene expression measurements
obtained from tissue samples to multivariate classi-
fication algorithms. Currently, published evidence on
clinical validity and utility for such assays as they are
offered to the patients is only available for MammaPrint
and OncotypeDx; for the remainder of these tests the
evidence derives from analyses performed in academic
settings [2].
* Correspondence: geman@jhu.edu; jleek@jhsph.edu
3
Institute for Computational Medicine, Johns Hopkins University, 3400 North
Charles Street, Baltimore, MD 21218, USA
2
Department of Biostatistics, Johns Hopkins Bloomberg School of Public
Health, 615 North Wolfe Street, Baltimore, MD 21205, USA
Full list of author information is available at the end of the article
Medicine (IOM), OncotypeDx was the most widely
used among these breast cancer assays, with more than
175,000 patients tested as of mid 2011, followed by
MammaPrint, used for 14,000 patients. OncotypeDX
combines the expression levels of 21 genes and was de-
veloped to predict the risk of distant recurrence at
10 years for women with lymph node negative, estrogen
receptor (ER) positive breast cancer [4]. MammaPrint
utilizes 70 genes to report a good or bad prognosis for
each patient, and was developed from microarray ex-
periments to predict 5-year metastatic recurrence of
breast cancer as a first event among ER positive and
negative patients [9,10]. The MammaPrint algorithm is
based on correlating the 70-gene expression profile of a
patient with a stored cancer profile in order to deter-
mine a risk score for the patient.
A relative small fraction of published cancer prog-
nostic markers have subsequently been introduced in
clinical practice, despite the large number of available
studies focusing on biomarkers development. A major

© 2013 Marchionni et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Marchionni et al. BMC Genomics 2013, 14:336
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hurdle hindering the translation of this research into
clinically useful assays has been identified in the lack of
rigorous criteria to report and publish tumor prognostic
marker studies [8]. This issue has been addressed by
introducing the REMARK guidelines, a set of recom-
mendations for tumor marker prognostic studies, which
provides the necessary framework for reporting all rele-
vant information about prognostic marker development
(i.e. study design, specimen and patient characteris-
tics, analytical and statistical methods) [11]. Another
key issue in the development of cancer biomarkers is
the need for detailed and complete disclosure of all
data and software [8,12,13]. This need is not specific
to the development of predictive signatures from
high-throughput molecular data but extends to many
other branches of computational medicine and biology
[14,15]. Whereas the guidelines for transparency in
genomic data sharing date back a decade to the adop-
tion of the Minimal Information About Microarray Ex-
periments (MIAMIE) standards [16], the recent scandal
leading to the decision to cancel three clinical trials
based on microarray-based gene expression screening
tests has dramatically underscored the need for revised
genomics research criteria [17] that extend and/or inte-
grate the REMARK and MIAME guidelines.
Maximizing the level of evidence on the spectrum of re-
producibility requires complete, independent replication
[18]. As measured by this criterion, neither of the two suc-
cessful breast cancer assays, MammaPrint and
OncotypeDX, provides a paradigmatic example of the way
genomic predictors should be developed. In the case of
OncotypeDX, the prediction algorithm is described in detail
and can be reprogrammed, but the original datasets used
for the implementation and validation [4] of the assay were
never placed in the public domain. Conversely, in the case
of MammaPrint, although the original discovery and valid-
ation datasets [3,19] are available, the pre-processing proto-
col and prediction algorithm are only partially described.
Thus the entire development, including data and code,
is not available for either MammaPrint nor OncotypeDX.
However, in the case of MammaPrint it is possible to
undertake a transparent re-analysis of the data using an al-
ternative approach, since the raw microarray data are
available. We therefore focus here our efforts on reprodu-
cing the results of Mammaprint. We collect and organize
the original MammaPrint discovery and validation data.
We also coordinate the associated metadata for these ex-
periments and develop reproducible documents for their
analysis. We reproduce and implement the preprocessing
described in the original manuscripts. These data repre-
sent a resource that can be used by other investigators
both to verify the original claims about the MammaPrint
signature and to build alternative predictors. As an ex-
ample of the utility of these data, we use the MammaPrint
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discovery and validation data to develop an alternative sig-
nature and prognostic test for breast cancer, which is
based on several two-gene comparisons [20,21]. This pro-
vides a detailed, transparent and fully reproducible ex-
ample of constructing a multi-gene classifier.
Methods
Data assembly and code
We collected the data from the original experiments used to
identify [9] and develop [10] the MammaPrint 70-gene
prognostic signature as provided as additional files with the
original manuscripts. We also collected from ArrayExpress
[22] the dataset used to retrain this signature on the custom
array currently used in the MammaPrint assay [3] as well as
the independent validation cohort using the same array [19].
All of these datasets have been organized in an open re-
source that can be used to develop and compare prognostic
signatures for breast cancer (available at http://
luigimarchionni.org/breastTSP.html) and Bioconductor
[23]. This resource also encompasses the R [24] code and
libraries used to retrieve, pre-process, manipulate, annotate,
and analyze these data. The code, fully annotated and exe-
cutable, is provided in the Additional files 1 and 2. All the
analyses performed in our study were based on de-identi-
fied publically available data, and they were performed in
compliance to the Helsinki declaration. The research did
not involve any experiment on human subjects or animals
and for this reason no ethical approval was necessary.
An example of reproducible signature development
In order to build our new classifier we selected the 78
patients originally used in the 70-gene prognostic signa-
ture discovery and limited our analysis to the 70 genes
contained in the original signature. We made these deci-
sions for two reasons: (a) to make our development
process entirely analogous to the process for
MammaPrint and (b) so that our signature can be calcu-
lated on the basis of the data from any current
MammaPrint assay. To this end it should also be noted
that the MammaPrint microarray platform only includes
the prognostic signature genes and a set of housekeeping
genes used for normalization purposes. These latter
genes are designed not to change across samples and
were therefore not used to train our predictor. We
adopted an extension of a rank-based approach to classi-
fication called “top-scoring pairs” (TSP) for developing
understandable and powerful genomic signatures. This
approach is invariant to all data preprocessing and
normalization steps that maintain the ordering within
sample gene expression profiles. The TSP algorithm se-
lects the pair of genes whose expression levels switch
their ranking most consistently between the two prog-
nostic groups (Figure 1). The original TSP algorithm
[20] and extensions [25] have previously been
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Figure 1 Top Scoring Pair. A Top Scoring Pair (TSP) is formed by a
pair of measurements that consistently change ranking between
samples from different prognostic groups.
successfully applied to differentiate [26], predict treat-
ment response in breast cancer [27] and acute myeloid
leukemia [28], and grade prostate cancers [29].
Building the K-TSP classifier
We recorded the relative ordering of each pair of
genes in the 70-gene MammaPrint signature in each of
the 78 training samples. In other words, for each pair
of genes g and g’, and for each sample j, we record
whether the expression of g in sample j is larger than
the expression of g’ in sample j or vice-versa. The “sig-
nature” for the TSP classifier is the pair of genes that
most consistently changes its relative expression order-
ing between the two groups of patients and the corre-
sponding decision rule for a new profile is determined
entirely by the ordering between these two genes:
choose group one if the observed ordering was most
often seen in group one and group two otherwise.
Here, the two groups of patients are those that re-
curred within 5 years (poor prognosis) and those that
who did not recur (good prognosis). The K-TSP algo-
rithm uses K pairs of genes. It proceeds by first identi-
fying the TSP, removing these two genes from the 70-
gene signature, then searching for the pair of genes
among the 68 remaining that most often switch their
ordering between groups, removing these from the list,
and so forth. Individually, each pair of genes “votes”
for one of the two groups based on the observed or-
dering. For a fixed number K of pairs, the final prog-
nostic score is the sum of the votes for the poor
prognosis group among all K pairs. The higher the
score, the more evidence there is for poor prognosis.
Selecting the number of pairs
For each possible number of pairs K we measured the
accuracy of the prognostic score on the training set by
calculating the area under the receiver operating charac-
teristic curve (AUC) [30] determined by considering all
possible score thresholds for declaring poor prognosis.
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Here we used re-substitution AUC for training, since the
TSP approach is based on binary decisions and is not
prone to overfitting. The AUC increased with K until
reaching a peak and then declined as further pairs were
added (Figure 2). We focused on values of K near the
peak AUC, namely K = 6 to K = 10, and only considered
score thresholds achieving 100% sensitivity. The number
of gene pairs K was then chosen to maximize specificity,
which is equivalent to choosing the maximum score
threshold which achieves 100% sensitivity. This resulted
in the 8-TSP classifier (Figure 3) with score threshold
two. Such resubstitution estimates obtained from the
training set of samples were used only for the model
optimization and do not reflect its performance, which
in turn was assessed on an independent cohort of pa-
tients (see below).
Validation of the 8-TSP signature in an independent
patients cohort
To evaluate the classifier on a new sample, the relative
ordering of each of the K = 8 pairs of genes is deter-
mined and the sample is assigned to the poor prognosis
group if there are two or more votes for poor prognosis
(Figure 3), using the same procedures previously defined
in the training set of patients. The 8-TSP signature and
the MammaPrint test were hence compared in terms of
classification performance, using standard measures
such as accuracy, sensitivity, specificity, and AUC, and
in term of survival, by Kaplan-Meier and Cox regression
analyses.
Results and discussion
We compared our prognostic test to the MammaPrint
test based on a large independent validation cohort
consisting of 307 patients from a European multi-center
Figure 2 Resubstittution performance in the training set.
Receiver Operator Characteristics (ROC) analysis was performed in
the training set and the Area Under the Curve (AUC) was used to
select the final number of TSPs. An 8-TSP classifier was chosen to
maintain 100% training set sensitivity and maximize specificity.
Marchionni et al. BMC Genomics 2013, 14:336
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Figure 3 8-TSP breast cancer prognosis signature. Each of the 8 gene pairs votes independently; patients with two or more votes are
classified as poor prognosis.
study [19]. In this independent validation cohort our test
achieved 91% sensitivity, 47% specificity, and 69% over-
all accuracy (Figures 4A and 4B, and Additional files 1
and 2). Sensitivity refers to correctly classifying poor
prognosis patients and specificity refers to correctly
classifying good prognosis patients. For comparison,
the MammaPrint prognostic test achieves 89% sensitivity,
42% specificity, and 65% overall accuracy [19,31] in this
same validation set. Such performance in predicting
metastatic recurrence within 5 years was reflected in the
AUC estimates: 0.69 (95% CI: 0.64 − 0.74) and 0.59 (95%
CI: 0.55 − 0.62) for the 8-TSP and MammaPrint respect-
ively. (Comparable results were obtained by PAM, a well-
known classification method; see the Additional files 1
and 2.) Finally, while in the prediction of a metastatic
event within five years the 8-TSP classifier performed
better than the MammaPrint test, this latter assay
maintained a better performance at later time points as
revealed in survival analyses. This finding probably indi-
cates that the additional features of the 70-gene signature
not used in the 8-TSP classifier might carry additional
prognostic information beyond five years (see Additional
files 1 and 2).
We have therefore built a prognostic classifier based
on the genes from the MammaPrint signature that is as
accurate in predicting 5-year disease-free survival as the
MammaPrint prognostic test based. Our classifier only
requires the measurement of expression for 16 of the 70
genes used in Mammaprint. Moreover, the new test is
easy to interpret and is robust with respect to any
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preprocessing of the expression data that maintains the
ordering among expression levels within sample profiles.
Finally, all design decisions and choices of parameters
were based entirely on the training set. There was no
“data leakage”: no test data was examined until all as-
pects of classifier development were “locked up.” These
are considered critical steps in developing reproducible
and accurate genomic signatures as defined by the IOM
report [8]. The two key parameters are K, the number of
pairs of genes in the signature, and the score threshold.
We only considered values of K between 6 and 10 since
these values maximized overall performance, and we
only considered thresholds that obtained 100% sensitiv-
ity. Under these design constraints, we selected the K = 8
since this value maximized specificity at 100% sensitivity
(Figure 2). Our final classifier labels a sample as poor
prognosis if two or more among the 8 pairs votes for the
poor prognosis group (Figure 3).
Our 8-TSP signature can be viewed as the combin-
ation of multiple coordinated biological processes. Of
the 70 genes originally identified in the study by van’t
Veer and colleagues [10], 18 genes had expression values
positively associated with good prognosis, while 52 were
associated with metastatic recurrence. Four of the K = 8
pairs combine genes positively correlated with good
prognosis (RTN4RL1, LGP2, MS4A7, and GSTM3) with
genes associated with bad prognosis (OXCT1, HRASLS,
Contig40831_RC, and MELK). These pairs represent a
coordinated change from good prognosis expression pat-
terns to poor prognosis patterns across multiple gene
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Figure 4 8-TSP classification results in the validation set. Panel

A) The 8-TSP results from the first 150 patients in the validation set.
Each column represents one of the 8 pairs (blue = good prognosis vote,
red = bad prognosis vote) and each row is a patient. Patients with bad
prognosis (top rows) have more votes for bad prognosis. Panel B) The
8-TSP results from the last 157 patients in the validation set.

pairs. The remaining pairs comprise only genes origin-

ally associated with a poor prognosis (GPR180, DTL,
IGFBP5, SERF1A, GNAZ, RFC4, CDCA7, and UCHL5),
suggesting that it is the quantitative level of expression
of these genes that is important for predicting prognosis.
It is of note that each individual TSP involved in the
final classification scheme can be viewed as a separate
molecular switch between the two prognostic groups,
possibly entailing also a mechanistic underpinning. To
this end some of the pairs we have identified appear to
have an additional underlying mechanistic biological re-
lationship. For instance one of the gene pairs, DTL-
RCF4, appears to be tightly associated with the regula-
tion of the replication fork and the DNA damage re-
sponse. DTL and RCF4 physically interact and modulate
the activity of the proliferating cell nuclear antigen
(PCNA) [32-34], which plays a central role in the coord-
ination of these processes. Similarly, another pair,
GPR180-GNAZ, code for proteins involved in G protein
mediated cellular signaling.

Conclusions
Our goal was to provide a transparent example of the
manner in which a genomics-based cancer predictor
might be developed from training data and evaluated on
independent test data with sufficient detail and docu-
mentation to allow the full process to be replicated by
other researchers. Due to the unavailability of the ori-
ginal data, it was not possible carry out this process for
OncotypeDX, which is presently the most used and vali-
dated predictor of this kind. Consequently, we
performed a re-analysis of MammaPrint data. To this
end, we selected the same samples and end-point origin-
ally used for the implementation of this assay, although
we are aware that a stratified analysis across ER positive
and negative patients would be much more appropriate.
In order to illustrate the development process from end
to end, including a transparent decision rule, we have in-
troduced a more parsimonious classifier with sensitivity,
specificity, and overall accuracy very similar to the 70-
gene MammaPrint signature.
Our analysis was performed in complete adherence to
the principles of transparent and reproducible research
[13,18], providing all data sources used, and the
complete code and software necessary for data prepro-
cessing, analysis and validation. To our knowledge, this
Marchionni et al. BMC Genomics 2013, 14:336
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is one of the few, if not the first, development of a gen-
omic signature adhering to these standards.
Additional files
Additional file 1: Fully reproducible vignette of the analysis.
Additional file 2: The archive contains the following files:
“bmc_article.bst”: BMC series bibliography style; “localFiles/
contactAgendia": instructions to obtain the hybridization mapping
information from Agendia; “objs/buyseEset.rda”: ExpressionSet for
the Buyse cohort; “objs/glasEset.rda”: ExpressionSet for the Glas
cohort; “Supplement.bib”: Bibliography in BibTex format;
“Supplement.Rnw”: Rnoweb/Sweave file containing code and text
used to create the “Supplement.tex” file; “Supplement.tex”: LaTeX
file resulting from running the Sweave with the “Supplement.Rnw”
file; All source code, data, and software packages used in the
analyses are also available for download online from: http://
luigimarchionni.org/breastTSP.html.
Abbreviations
TSP: Top scoring pair; ROC: Receiver operator curve; AUC: Area under the
curve; FDA: Food and drug administration; IOM: Institute of medicine;
ER: Estrogen receptor; REMARK: Reporting recommendations for tumour
marker prognostic studies; MIAMIE: Minimal information about microarray
experiments; CI: Confidence intervals; RTN4RL1: Reticulon 4 receptor-like 1;
LGP2: DHX58 DEXH (ASP-GLU-X-HIS) box polypeptide 58; MS4A7: MS4A7
membrane-spanning 4-domains, subfamily A, member 7; GSTM3: Glutathione
S-Transferase MU 3 (BRAIN); OXCT1: 3-oxoacid coa transferase 1;
HRASLS: HRAS-Like suppressor; MELK: Maternal embryonic leucine zipper
kinase; GPR180: G Protein-coupled receptor 180; DTL: Denticleless E3
ubiquitin protein ligase homolog (drosophila); IGFBP5: Insulin-like growth
factor binding protein 5; SERF1A: Small edrk-rich factor 1A (TELOMERIC);
GNAZ: Guanine nucleotide binding protein (G protein), alpha Z polypeptide;
RFC4: Replication factor C (activator 1) 4, 37KDA; CDCA7: Cell division cycle
associated 7; UCHL5: Ubiquitin carboxyl-terminal hydrolase L5;
PCNA: Proliferating cell nuclear antigen.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LM, JTL, and DG conceived the study; LM performed all the analysis and
assembled all the datasets; LM and BA implemented the software packages
used in the analysis; LM, JTL and DG wrote the manuscript. All authors read
and approved the manuscript.
Acknowledgements
The authors express gratitude to Antonio C. Wolff for the invaluable
comments, and Annuska M Glas for the information on the datasets.
Funding
This work was supported by the Johns Hopkins Breast Cancer Program through
funding from the Safeway Research Foundation, and by the National Institute of
Health (P30 CA006973 to LM, and R01 GM08308 to JTL).
Author details
1 Bioinformatics 2009, 25(9):1203–1204.
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University
School of Medicine, 1550 Orleans Street, Baltimore, MD 21231, USA.
2 and analysis in ArrayExpress. Methods Enzymol 2006, 411:370–386.
Department of Biostatistics, Johns Hopkins Bloomberg School of Public
Health, 615 North Wolfe Street, Baltimore, MD 21205, USA. 3Institute for
Computational Medicine, Johns Hopkins University, 3400 North Charles
Street, Baltimore, MD 21218, USA. 4Department of Applied Mathematics and
Statistics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD
21218, USA. 5Center for Computational Biology, Johns Hopkins University,
Baltimore, MD 21205, USA.
Received: 18 December 2012 Accepted: 4 May 2013
Published: 17 May 2013
Page 6 of 7
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doi:10.1186/1471-2164-14-336
Cite this article as: Marchionni et al.: A simple and reproducible breast
cancer prognostic test. BMC Genomics 2013 14:336.

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